Lóránt Király

Suppression of tobacco mosaic virus-induced hypersensitive-type necrotization in tobacco at high temperature is associated with downregulation of NADPH oxidase and superoxide and stimulation of dehydroascorbate reductase

Tissue necroses and resistance during the hypersensitive response (HR) of tobacco to tobacco mosaic virus (TMV) are overcome at temperatures above 28 degrees C and the virus multiplies to high levels in the originally resistant N-gene expressing plants. We have demonstrated that chemical compounds that generate reactive oxygen species (ROS) or directly applied hydrogen peroxide (H(2)O(2)) are able to induce HR-type necroses in TMV-inoculated Xanthi-nc tobacco even at high temperatures (e.g. 30 degrees C). The amount of superoxide (O(2)(*-)) decreased, while H(2)O(2) slightly increased in TMV- and mock-inoculated leaves at 30 degrees C, as compared with 20 degrees C. Activity of NADPH oxidase and mRNA levels of genes that encode NADPH oxidase and an alternative oxidase, respectively, were significantly lower, while activity of dehydroascorbate reductase was significantly higher at 30 degrees C, as compared with 20 degrees C. It was possible to reverse or suppress the chemically induced HR-type necrotization at 30 degrees C by the application of antioxidants, such as superoxide dismutase and catalase, demonstrating that the development of HR-type necroses indeed depends on a certain level of superoxide and other ROS. Importantly, high TMV levels at 30 degrees C were similar in infected plants, whether the HR-type necrotization developed or not. Suppression of virus multiplication in resistant, HR-producing tobacco at lower temperatures seems to be independent of the appearance of necroses but is associated with temperatures below 28 degrees C.

Uncoupling Resistance from Cell Death in the Hypersensitive Response of Nicotiana Species to Cauliflower mosaic virus Infection

Cauliflower mosaic virus strain W260 elicits a hypersensitive response (HR) in leaves of Nicotiana edwardsonii, an interspecific hybrid derived from a cross between N. glutinosa and N. clevelandii. Interestingly, we found that N. glutinosa is resistant to W260, but responds with local chlorotic lesions rather than necrotic lesions. In contrast, N. clevelandii responds to W260 with systemic cell death. The reactions of the progenitors of N. edwardsonii to W260 infection indicated that each contributed a factor toward the development of HR. In this study, we present two lines of evidence to show that the resistance and cell death that comprise the HR elicited by W260 can indeed be uncoupled. First, we showed that the non-necrotic resistance response of N. glutinosa could be converted to HR when these plants were crossed with N. clevelandii. Second, we found that cell death and resistance segregated independently in the F2 population of a cross between N. edwardsonii and N. clevelandii. We concluded that the resistance of N. edwardsonii to W260 infection was conditioned by a gene derived from N. glutinosa, whereas cell death was conditioned by a gene derived from N. clevelandii. An analysis of pathogenesis-related (PR) protein expression in response to W260 infection revealed that elicitation of PR proteins was associated with resistance rather than with the onset of cell death.

Systemic Cell Death Is Elicited by the Interaction of a Single Gene in Nicotiana clevelandii and Gene VI of Cauliflower Mosaic Virus

Cauliflower mosaic virus (CaMV) strains D4 and W260 can be distinguished by the type of symptoms they induce in Nicotiana clevelandii and N. edwardsonii. W260 induces systemic cell death in addition to a mosaic symptom in N. clevelandii and a hypersensitive response (HR) in N. edwardsonii, whereas D4 induces a systemic mosaic in both hosts. To determine which W260 genes are responsible for systemic cell death, chimeric viruses were constructed between the D4 and W260 strains. It was found that W260 gene VI was responsible for the elicitation of systemic cell death; previous studies had shown that this same gene elicited HR in N. edwardsonii. An immunological analysis of plants infected with W260 or D4 indicated that the systemic cell death symptom was not associated with enhanced levels of either W260 virions or the W260 gene VI product. To investigate the inheritance of systemic cell death, crosses were made between N. clevelandii and N. bigelovii, a host that reacts with a systemic mosaic symptom upon i...

Functional assessment of the pathogenesis-related protein PR-1b in barley

Abstract The pathogenesis-related protein 1 (PR-1b) of barley (Hordeum vulgare L.) is a marker for the attack by the powdery mildew fungus (Blumeria graminis f.sp. hordei, Bgh) and other pathogens. PR-1b consists of 164 amino acids and has a potential signal peptide for export into the cell wall. Here, we show that PR-1b is differentially expressed in near-isogenic barley lines exhibiting various forms of defence phenotypes including papilla formation and the hypersensitive cell death. To elucidate PR-1b function, we transiently silenced PR-1 expression by double stranded RNA (dsRNA) interference in the moderately susceptible barley double mutant line A89 (genotype: mlo5–ror1), which shows a papillae-based defence phenotype. Upon bombardment of leaf segments with PR-1b dsRNA and a GFP marker gene construct, Bgh slightly more frequently penetrated the plant cell wall of transformed epidermal cells relative to cells bombarded with human control dsRNA. We conclude that PR-1b contributes to penetration resistance to the powdery mildew fungus in barley. We also observed that PR-1b expression correlates with the production of H2O2 in responses to Bgh and Bipolaris sorokiniana and was induced upon infiltration of the H2O2 producing mixture of glucose and glucose oxidase.

Sulfate supply influences compartment specific glutathione metabolism and confers enhanced resistance to Tobacco mosaic virus during a hypersensitive response

Sufficient sulfate supply has been linked to the development of sulfur induced resistance or sulfur enhanced defense (SIR/SED) in plants. In this study we investigated the effects of sulfate (S) supply on the response of genetically resistant tobacco (Nicotiana tabacum cv. Samsun NN) to Tobacco mosaic virus (TMV). Plants grown with sufficient sulfate (+S plants) developed significantly less necrotic lesions during a hypersensitive response (HR) when compared to plants grown without sulfate (−S plants). In +S plants reduced TMV accumulation was evident on the level of viral RNA. Enhanced virus resistance correlated with elevated levels of cysteine and glutathione and early induction of a Tau class glutathione S-transferase and a salicylic acid-binding catalase gene. These data indicate that the elevated antioxidant capacity of +S plants was able to reduce the effects of HR, leading to enhanced virus resistance. Expression of pathogenesis-related genes was also markedly up-regulated in +S plants after TMV-inoculation. On the subcellular level, comparison of TMV-inoculated +S and −S plants revealed that +S plants contained 55–132 % higher glutathione levels in mitochondria, chloroplasts, nuclei, peroxisomes and the cytosol than −S plants. Interestingly, mitochondria were the only organelles where TMV-inoculation resulted in a decrease of glutathione levels when compared to mock-inoculated plants. This was particularly obvious in −S plants, where the development of necrotic lesions was more pronounced. In summary, the overall higher antioxidative capacity and elevated activation of defense genes in +S plants indicate that sufficient sulfate supply enhances a preexisting plant defense reaction resulting in reduced symptom development and virus accumulation.

The Plant Gene CCD1 Selectively Blocks Cell Death During the Hypersensitive Response to Cauliflower Mosaic Virus Infection

The P6 protein of Cauliflower mosaic virus (CaMV) W260 elicits a hypersensitive response (HR) on inoculated leaves of Nicotiana edwardsonii. This defense response, common to many plant pathogens, has two key characteristics, cell death within the initially infected tissues and restriction of the pathogen to this area. We present evidence that a plant gene designated CCD1, originally identified in N. bigelovii, can selectively block the cell death pathway during HR, whereas the resistance pathway against W260 remains intact. Suppression of cell death was evident not only macroscopically but also microscopically. The suppression of HR-mediated cell death was specific to CaMV, as Tobacco mosaic virus was able to elicit HR in the plants that contained CCD1. CCD1 also blocks the development of a systemic cell death symptom induced specifically by the P6 protein of W260 in N. clevelandii. Introgression of CCD1 from N. bigelovii into N. clevelandii blocked the development of systemic cell death in response to W260 infection but could not prevent systemic cell death induced by Tomato bushy stunt virus. Thus, CCD1 blocks both local and systemic cell death induced by P6 of W260 but does not act as a general suppressor of cell death induced by other plant viruses. Furthermore, experiments with CCD1 provide further evidence that cell death could be uncoupled from resistance in the HR of Nicotiana edwardsonii to CaMV W260.

Up-Regulation of Antioxidants in Tobacco by Low Concentrations of H 2 O 2 Suppresses Necrotic Disease Symptoms

Pretreatment of tobacco leaves with low concentrations (5 to 10 mM) of H₂O₂ suppressed hypersensitive-type necrosis associated with resistance to Tobacco mosaic virus (TMV) or Pseudomonas syringae pv. phaseolicola. The same pretreatment resulted in suppression of normosensitive necrosis associated with susceptibility to Botrytis cinerea. This type of H₂O₂-mediated, induced disease symptom resistance correlated with enhanced host antioxidant capacity, i.e., elevated enzymatic activities of catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (POX) after viral and bacterial infections. Induction of genes that encode the antioxidants superoxide dismutase (SOD), CAT, and APX was also enhanced early after TMV infection. Artificial application of SOD and CAT suppressed necroses caused by viral, bacterial, or fungal pathogens similarly as H₂O₂ pretreatment, implying that H₂O₂-mediated symptom resistance operates through enhancement of plant antioxidant capacity. Pathogen multiplication was not significantly affected in H₂O₂-pretreated plants. Salicylic acid (SA), a central component of plant defense, does not seem to function in this type of H₂O₂-mediated symptom resistance, indicated by unchanged levels of free and bound SA and a lack of early up-regulation of an SA glucosyltransferase gene in TMV-infected H₂O₂-pretreated tobacco. Taken together, H₂O₂-mediated, induced resistance to necrotic symptoms in tobacco seems to depend on enhanced antioxidant capacity.

Enhanced Inducibility of Antioxidant Systems in a Nicotiana tabacum L. Biotype Results in Acifluorfen Resistance

Abstract Levels of non-protein thiols (mostly glutathione, GSH), ascorbic acid (AA), and activities of the enzymes ascorbate peroxidase (AP), glutathione reductase (GR) and GSH S-transferase (GST) were determined in cell-free leaf extracts of acifluorfen-resistant and -sensitive tobacco plants. These parameters were examined also in detached leaves of the above plants exposed to acifluorfen stress. In leaves of untreated plants the AA content was by 40% higher in the resist ant biotype as compared to the sensitive ones, but the levels of GSH, AP, GR, and GST did not differ significantly in the two biotypes. However, in the resistant leaves stressed by acifluorfen the activity of AP readily increased while in the sensitive leaves it did not change. The levels of GSH and the activities of GR and GST markedly increased in both biotypes after acifluorfen stress, but the induction in the resistant leaves was consistently stronger in each case. The AA contents were increased equally in both biotypes. These parameters were much less affected by paraquat stress. The only significant changes were observed at low concentrations of this herbicide (8 x 10-9 м): when the thiol content and the activity of GST increased in the resistant leaves. Enhanced inducibility of antioxidant systems seems to be involved in resistance of tobacco to acifluorfen stress.

Temporal and Spatial Appearance of Recombinant Viruses Formed Between Cauliflower Mosaic Virus (CaMV) and CaMV Sequences Present in Transgenic Nicotiana bigelovii

Cauliflower mosaic virus (CaMV) strain CM1841 is able to recombine with a CaMV transgene sequence present in Nicotiana bigelovii. In the present study we have characterized the temporal and spatial appearance of recombinant viruses formed between CM1841 and the transgene within individual transgenic plants. CM1841 infections were initiated by mechanical inoculation and by agro-inoculation to nontransformed N. bigelovii and transgenic N. bigelovii that expressed the gene VI product of CaMV strain D4. In agroinoculated transgenic plants, inoculated leaf tissue turned necrotic around the point of agroinocu-lation, while chlorotic lesions appeared in the leaves inoculated with CM1841 virions. The first systemic symptoms in both agroinoculated and mechanically inoculated transgenic N. bigelovii consisted of necrotic patches. The predominant type of virus recovered from the inoculated and first systemically infected leaves was the wild-type CM1841 rather than a recombinant. As the infection progressed in the tr...

Reactive Oxygen Species and Plant Disease Resistance

Plants may successfully limit or even kill pathogens at least in part by eliciting spatial patterns of ROS production in different parts of invaded plant cells, e.g., the cell wall and plasma membrane. Recent research also suggests a significant contribution to plant disease resistance by ROS-mediated processes in the plant cuticle and intracellular organelles. The role of temporal patterns (i.e., proper timing) of ROS accumulation in eliciting an effective plant disease resistance is also discussed. Essentially, defense against pathogens could be very effective if it is a rapid, symptomless process, eliminating the pathogen in due time and not overusing resources of the plant, a process likely mediated by ROS. On the other hand, a delayed and failed attempt by the host to elicit resistance may result in massively stressed plant tissues and a partial or almost complete loss of control over pathogen invasion. Thus, it seems that when plants encounter pathogens they need to defend themselves simultaneously against biotic and abiotic stresses (i.e., pathogen accumulation and excessive cell/tissue death) by turning on two different types of—partially overlapping—signaling pathways that may function in parallel. Very recent interesting data suggest a pivotal role of autopropagating ROS waves in these signaling processes.